Molecular tagging of biomolecules provides an indispensable tool used in many fields such as biochemistry, molecular biology, immunology and medicine. Radioiodinated bio-ligands and bio-macromolecules are the most abundant examples of molecular tagging. Because of its high specific activity, significant half life and relatively simple preparative procedures radioiodinations are among the most frequent labeling approaches employed.
The high specific activity and the significant half life of .sup.125 I makes this isotope especially suitable for labeling and tracing of minute amounts of biomolecules. Some of the major research areas which rely heavily on radioiodinated preparations include receptor studies, affinity labeling and immunochemistry. The methods available today for radioiodination include direct methods where in situ oxidation of .sup.125 I by a variety of oxidants is carried out in the presence of the compound being subjected to radioiodination, and indirect methods where pre-radioiodinated reagents are used to N-modify amino functions in compounds of interest.
Some of the methods of direct radioiodinations include chloramine T (N-chloro-4-methylbenzensulfonamide sodium salt) (See Greenwood, F. C. et al., Biochem. J. 89, (1963)), Iodo-Beads (polymeric chloramine T) (See Markwell, M.A.K., Anal Biochem. 125, (1983), Iodo-Gen (1,3,4,6-tetrachloro-3a-6a-diphenylglycoluril) (See Fraker, P. J. et al., Biochem. Biophys. Res. Commun. 80, (1987)), Lactoperoxidase (See Thorell, J. I. et al., Biochim. Biophys. Acta 25, (1971)), and electochemical oxidation (Teare, F. W. et al., Intl. J. Appl. Rad. Isot. 29, (1978)). These radioiodinations occur on aromatic moieties such as phenolic, imidazolyl and indolyl which are sufficiently active toward electrophilic substitution. In general, these oxidative methods lead to complex reaction mixtures containing radioactive components (See Koshland, M. E. et al. J. Biol. Chem. 238, (1963)).
Polyiodinations, oxidations, reductions (when reducing agents such as sodium metabisulfite are used to decompose excess of the oxidizing reagent) and the presence of multiple reactive moieties in a single biomolecule accompanied by the lack of sufficient selectivity of the radioiodinating reagent, result in heterogeneous preparations which require tedious purifications.
The indirect radioiodination employs pre-labeled reagents thus avoiding the damage caused by the direct iodinations (See Bolton, A. E. et al., Biochem. J. 133, (1973) and Wood, F. T. et al., Anal. Biochem. 69, (1975)). To date only the mild acylating reagent N-succinimidyl 3-(4-hydroxy, 5-[.sup.125 I]iodophenyl) propionate (known as the Bolton-Hunter reagent) is used for achieving non-oxidative indirect radioiodinations (See Bolton, A. E. et al., Biochem. J. 133, (1973)). The Bolton-Hunter reagent acylates predominantly primary .epsilon.-amino functions of lysine residues and to a lesser extend N-terminal .alpha.-amino functions. In spite of the mild conditions under which the N-acylation by Bolton-Hunter reagent occurs, the heterogeneity of radioiodinated product results from the high abundance of multiple lysine residues in peptides and proteins which leads to hetero-mono and hetero-poly radioiodinated tracer (See Bolander, Jr. F. F. et al., Biochem. 14, (1975)). Furthermore, the susceptibility of the N-succinimidyl ester in the Bolton-Hunter reagent to hydrolysis limits it shelflife and calls for introduction of large molar excess of substrate to achieve efficient incorporations. This has obvious disadvantages when the substrate for labeling is a material which is hard to obtain. Under forcing conditions, where excess of Bolton-Hunter reagent is employed, acylation of histidine and tyrosine residues may also occur (See Knight, L. C., Biochim. Biophys. Acta 534, (1978)).
It was, therefore, an object of this invention to develop an indirect, mild and highly selective radiolabeling method which combines the advantage of the high specificity of the maleimido moiety towards a sulfhydryl group which results in an efficient and quantitative addition of thiols across the activated double bond of the maleimido moiety to form a stable thio-ether. The specificity of this reaction coupled with both the low abundance of cysteine residues in many proteins and bioactive peptides, and the ease of introduction of a cysteine residue or thiol containing moiety into synthetic peptide analogs allows for selective and specific iodination. Based on the low abundance of cysteine in peptides and the specificity and high reactivity of a sulfhydryl function toward a maleimido moiety, it was an object of this invention to develop a novel approach to indirect radiolabeling of peptides containing sulfhydryl groups by using the maleimido-based reagents of this invention.